We have used 9 synthetic peptides corresponding to sequences of polyoma virus small-T, middle-T and large-T antigens as immunogens in order to map antigenic epitopes that can induce polyoma-tumor-specific immunity in different mouse strains. We found that immunization of mice with synthetic peptides derived from amino acid (aa) sequences common to all 3 T-antigens (aa 1-19), or sequences common to only middle-T and small-T (aa 162-176), as well as synthetic peptides unique for middle-T (aa 269-282 and 371-381), or unique for large-T (aa 108-124, 316-333 and 436-449) can induce immunity against polyoma tumors. The synthetic peptides can be divided into 3 types with regard to immunogenicity; (i) peptides that immunize in more than one mouse strain and may represent immunodominant sites, (ii) peptides that may be immunogenic in only one strain, and thus strain-specific, and finally (iii) peptides that do not immunize in the strains tested so far.
Viral and tumor antigens are presented to cytotoxic T cells (CTL) in the form of short peptides. The peptide antigen is transported to the cell surface in conjunction with molecules encoded by the major histocompatibility complex (MHC). Different methods have recently been described for the analysis of the MHC-class-I binding ability of synthetic peptides. Here, we describe a protocol, based on intact RMA-S cells cultured at 26 degrees C in the presence of synthetic peptides, which gives an allele-specific peptide binding pattern with high resolution. This allowed an analysis of the H-2Kb-, Db- and Dd-binding capacity of a panel of synthetic peptides with amino acid (aa) sequences derived from polyoma (py) virus large-, middle- and small-T (LT, MT and ST) antigens, previously used in immunization experiments against py-virus-induced tumors. Eight single aa mutants and a deletion mutant of one peptide with an ability to bind both to H-2Kb and to Db were also analyzed. We foresee that the present protocol, or variants thereof, may serve as a simple and rapid assay for the systematic screening of the class-I binding ability of large sets of synthetic peptides in vitro. Such analysis may facilitate the search for viral or tumor peptide epitopes that are recognized by CTL.
An immunodominant polyoma peptide antigen MT162-176 was modified with regard to amino acid (aa) composition in an attempt to analyze its immunogenicity in detail. Twelve modifications of peptide MT162-176, 3 overlapping peptides and 9 peptides with point mutations, were synthesized and used for immunizations of (A.CA x C57BL/6)F1 and CBA mice against the syngeneic polyoma tumors SECA and SEBA. All 3 overlapping peptides MT162-176, MT165-174 and MT170-176, were immunogenic in (A.CA x C57BL/6)F1 mice against SECA, indicating that possibly more than one immunogenic epitope may be recognized within the MT162-176 sequence. In CBA mice, the 2 peptides covering the C-terminal half were immunogenic against SEBA, while the N-terminal peptide was possibly somewhat less efficient. The peptides with aa point mutations induced different anti-tumor responses in the 2 mouse strains. In CBA mice, only one mutant, MT162-176.28, was immunogenic. For (A.CA x C57BI)F1 3 different mutants, MT162-176.29, MT162-176.35 and MT162-176.36 were immunogenic against SECA, while the remaining 6 had lost their activity. These results suggest that a different emphasis of recognition of peptide MT162-176 exists with regard to the 2 mouse strains examined. Furthermore, different immunization procedures were tested. We found that repeated immunizations with peptide without Freund's adjuvant was the most efficient.
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